Method of enhancing expression of exogenous polynucleotide sequences in plants

ABSTRACT

A method of enhancing an expression of an exogenous polynucleotide sequence in a plant which includes administering to the plant a virus selected capable of suppressing gene silencing in the plant, thereby enhancing the expression of the transgene in the plant.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to a method of suppressing gene silencingin plants expressing exogenous polynucleotide sequences and, moreparticularly, to the use of infective avirulent virus strains forenhancing expression of exogenous polynucleotide sequences in plants.

The use of genetically modified crop is growing rapidly; at present,commercially-grown transgenic crops include soybean, corn, cotton, andcanola, which are cultivated in over 100 million acres worldwide. Suchgenetically modified commercial cultivars express exogenouspolynucleotide sequences which encode herbicide tolerance, insectresistance, virus resistance, male sterility, modified color, delayedripening and altered oil content.

In an emerging industry generically known as “molecular farming”,genetically modified plants are being used for the production ofcommercially valuable biomolecules such as polypeptides. Among theapplications that are currently being developed in molecular farming arethe production of low-cost antibodies for therapeutic and diagnosticuses, the production of copious amounts of hormones, cytokines and otherbio-active molecules for the treatment of chronic or lethal diseases,the production of bio-safe substitutes for various blood components, theproduction of degradable plastic biopolymers, the production ofunlimited amounts of processing enzymes for the food and pulp industry,the production of low-cost enzymes for waste treatments, and theproduction of safe bio-active molecules for the cosmetic industry.

Although useful in generating commercial amounts of such biomolecules,genetically modified plants, and in particular those that stably carrythe exogenous polynucleotide sequences in their genome (also referred toas transgenic plants) suffer from one major limitation which can limittheir ability to generate a commercial amount of the expressedbiomolecules. Shortly after the introduction of transgenic plants, itwas observed that transgene expression is at times downregulated, aphenomenon now known as “gene silencing” (Wassenegger and Pelissier,Plant Mol. Biol. 37:349-362, 1998; Napoli et al., Plant Cell 2: 279-289,1990; van der Krol et al., Plant Cell 2:291-299, 1990). There are twokinds of known gene silencing mechanisms: transcriptional gene silencing(TGS), which results from promoter inactivation and post-transcriptionalgene silencing (PTGS) which occurs when the promoter is active but themRNA fails to accumulate (Stam et al., Annals of Botany 79: 3-12, 1997).The gene-silencing phenomenon is particularly problematic in cases wherehigh level of transgenic expression is desired, such as the case inmolecular farming. Thus, to fully exploit molecular farming potential,gene silencing needs to be controlled.

Gene silencing is believed to evolve in plants as a defense mechanismagainst viral infection. However, plant viruses also have evolved toavoid or suppress the host gene silencing response. Moreover, in aseemingly contradictory manner, viruses can both induce and suppressgene silencing in plants (Marathe et al., 2000; Vaucheret et al., 2001).For example, co-inoculation of a normally innocuous virus with apotyvirus (e.g., tobacco etch virus, potato virus Y) leads tosubstantially increasing the disease severity caused by the otherwiseinnocuous virus. It was further demonstrated that the co-infectionsynergy resulted from suppressing gene silencing by a HC protease(HC-Pro) encoded by the potyvirus (Pruss et al. 1997). Later studiesreported that HC-Pro that was expressed in potyvirus-infected plantsalso suppressed gene silencing in transgenic plants (Anandalakshmi etal. 1998; Kasschau and Carrington, 1998; and Savenkov and Valkonen,2001). A recent survey revealed that gene silencing may be suppressed bymany different virulent viruses (Voinnet et al., 1999).

The present invention provides a novel approach for enhancing expressionof exogenous polynucleotide sequences in commercial plants byinoculating the plants with a selected virus which is avirulent yetcapable of suppressing gene silencing in the plants.

SUMMARY OF THE INVENTION

According to one aspect of the present invention there is provided amethod of enhancing an expression of an exogenous polynucleotidesequence in a plant. The method includes administering to the plant avirus selected capable of suppressing gene silencing in the plant,thereby enhancing the expression of the exogenous polynucleotidesequence in the plant.

According to another aspect of the present invention there is provided amethod of producing a molecule of interest. The method includesadministering to a plant a virus selected capable of suppressing genesilencing in the plant followed by extracting the molecule of interestbeing expressed in the plant, thereby producing the molecule ofinterest.

According to yet another aspect of the present invention there isprovided a method of identifying a gene-silencing agent. The methodincludes inoculating a plurality of transgenic plants with a pluralityof virus isolates or strains followed by selecting a plant from theinfected plants which exhibits a substantially higher level of exogenouspolynucleotide sequence expression than a non-infected similartransgenic plant, thereby identifying the virus isolate or straininfecting the plant as the gene silencing agent.

According to an additional aspect of the present invention there isprovided an article-of-manufacturing that comprises a containerincluding a virus selected capable of suppressing gene silencing in aplant, and a packaging material identifying the virus for use ininnoculating the plant.

According to further features in preferred embodiments of the inventiondescribed below, the virus is a systemically infectious virus.

According to still further features in the described preferredembodiments the virus is an avirulent virus.

According to still further features in the described preferredembodiments the virus is a mechanically transmitted virus.

According to still further features in the described preferredembodiments the administering is effected by using an inoculation gun.

According to still further features in the described preferredembodiments the step of inoculating a plurality of transgenic plantswith a plurality of virus isolates or strains further includes selectingplants which do not exhibit severe symptoms.

According to still further features in the described preferredembodiments the symptoms are selected from the group consisting ofmosaic, ring spots, leaf roll, yellowing, streaking, pox formation,tumor formation, pitting and stunting.

According to still further features in the described preferredembodiments the step of inoculating a plurality of transgenic plantswith a plurality of virus isolates or strains is effected byadministering the virus isolates or strains suspended in a buffersolution supplemented with an abrasive material onto foliage of thetransgenic plants.

According to still further features in the described preferredembodiments the exogenous polynucleotide sequence expression isquantified by an exogenous polynucleotide sequence transcribed mRNAlevel.

According to still further features in the described preferredembodiments the exogenous polynucleotide sequence expression isquantified by an exogenous polynucleotide sequence encoded polypeptidelevel.

According to still further features in the described preferredembodiments the molecule of interest is selected from the groupconsisting of an antibody, a vaccine, a therapeutic polypeptide, anindustrial enzyme and a biopolymer.

According to still further features in the described preferredembodiments the molecule of interest is a polypeptide capable ofconferring resistance or tolerance to biotic stress.

According to still further features in the described preferredembodiments the molecule of interest is a polypeptide capable ofconferring resistance or tolerance to abiotic stress.

According to still further features in the described preferredembodiments the molecule of interest is a nutritionally valuablepolypeptide.

According to still further features in the described preferredembodiments the virus is lyophilized.

The present invention successfully addresses the shortcomings of thepresently known configurations by providing a method of enhancingexpression of exogenous polynucleotide sequences in plants by infectingthe plants with viruses capable of suppressing gene silencing in theplants.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice.

In the drawings:

FIG. 1 is a quantitative fluorometer analysis illustrating the effect ofPVY strain N605 infection on the expression of an exogenouspolynucleotide sequence encoded GFP in Nicotiana tabacuum.A=non-infected wild type control; B=virus-infected wild type;C=non-infected transgenic plant; D=virus-infected transgenic plant.

FIG. 2 is a quantitative Northern blot analysis illustrating the effectof CMV Banana strain infection on the expression of an exogenouspolynucleotide sequence transcribed mRNA in Arabidopsis thaliana. Lane1=non-infected wild type control (19,100); Lane 2=virus-infectedtransgenic plant (765,700); Lane 3=non-infected transgenic plant(320,300); Lane 4=virus-infected wild type (135,500); Lane5=virus-infected transgenic plant (715,000).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is of a method of enhancing expression ofexogenous polynucleotide sequences in plants which method is based oninfecting the plants with an avirulent virus selected capable ofsuppressing gene silencing in the plants.

The principles and operation of the present invention may be betterunderstood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details of construction and the arrangement of the components setforth in the following description or illustrated in the drawings. Theinvention is capable of other embodiments or of being practiced orcarried out in various ways. Also, it is to be understood that thephraseology and terminology employed herein is for the purpose ofdescription and should not be regarded as limiting.

While reducing the present invention to practice, the present inventorsdemonstrated for the first time that selected virus strains which arecapable of systemically infecting transgenic plants without causingsevere symptoms, are also capable of enhancing expression of exogenouspolynucleotide sequences stably integrated into a genome of the infectedplants (see Examples 1 and 2 hereinbelow for further detail).

Although the use of isolated viral sequences in downregulating genesilencing in plants and thus enhancing expression of transgenes has beenpreviously suggested (see, U.S. Pat. Nos. 5,939,541; 6,395,962;5,939,541 and 6,395,962), use of such isolated sequences requirestransforming plants with viral components and, therefore, it is notapplicable for treating existing commercial cultivars. In addition,developing new transgenic cultivars is always a laborious andtime-consuming process, which may not be commercially worthwhile.Furthermore, the introduction of a viral component to plants may notnecessarily mimic the effect of viral infection on gene silencing.

Thus, according to one aspect of the present invention, there isprovided a method of enhancing an expression of an exogenouspolynucleotide sequence in a plant by administering to the plant a virusselected capable of enhancing the expression of the exogenoiuspolypeptide by preferably suppressing the gene silencing mechanism inthe plant.

As used herein, the term “suppressing” when used with respect to genesilencing refers to partial or complete inhibition of at least onecomponent of the gene silencing mechanism.

As used herein, the term “exogenous polynucleotide sequence” refers toany nucleic acid sequence which does not naturally occur within theplant but which, when introduced into the plant either in a stable ortransient manner, produces a polypeptide product. According to preferredembodiments of the present invention, the exogenous polynucleotidesequence is stably integrated into the plant genome in which case, theplant is typically referred to as a transgenic plant. The exogenouspolynucleotide sequence can encode any type of polypeptide expressiblein plants. Examples of polypeptides which are expressible in plants areprovided hereinbelow.

Plant-derived polypeptides are cheaper to produce and store, easier toscale up for mass production, and safer than those derived from animalsor microorganisms. Recent reviews of molecular farming include Daniellet al., (Medical molecular farming: production of antibodies,biopharmaceuticals and edible vaccines in plants, Trends Plant Sci.6:219-226, 2001), Rishi et al. Molecular Farming in Plants: A CurrentPerspective, J. Plant Biochemistry and Biotechnology 10:1-12, 2001),Mor, et al. (Edible vaccines: a concept comes of age, Trends Microbiol.6:449-453, 1998) and Tacket and Mason (A review of oral vaccination withtransgenic vegetables, Microbes Infect. 1:777-783, 1999).

Polypeptides with applications in human or animal vaccines which can beexpressed by transgenic plants currently include enterotoxigenic E. colivaccine expressed in tobacco, potato and maize; Vibrio cholerae vaccineexpressed in potato; Hepatitis B virus vaccine expressed in tobacco,potato, lupin and lettuce; Norwalk virus vacrine expressed in tobaccoand potato; Rabies virus vaccine expressed in tomato; humancytomegalovirus vaccine expressed in tobacco; Rabbit hemorrhagic diseasevirus vaccine, expressed in potato; foot-and-mouth disease vaccineexpressed in Arabidopsis and alfalfa; and transmissible gastroenteritiscoronavirus expressed in Arabidopsis, tobacco and maize (Daniell et al.,Trends Plant Sci. 6:219-226, 2001).

Antibodies expressed by transgenic plants currently include antiglycoprotein B of HSV, expressed in soybean; anti colon cancer markerantibody, expressed in tobacco; anti S. mutans (tooth decay) antibody,expressed in tobacco; Hodgkin's lymphoma ScFv of IgG from mouse,expressed in tobacco; B-cell lymphoma antibody, expressed in tobacco;anti carcinoembryogenic marker ScFV, expressed in cereals; anti humancreatine kinase, expressed in Arabidopsis; anti Atrazine antibody,expressed in tobacco; and anti nematode antigen, expressed in tobacco(Rishi et al., J Plant Biochemistry and Biotechnology 10:1-12, 2001).

Polypeptides with applications in human health which can be expressed bytransgenic plants currently include human protein C, expressed intobacco; human hirudin, expressed in canola (Brassica napus); humangranulocyte-macrophage colony-stimulating factor, expressed in tobacco;human somatotropin, expressed in tobacco; Nuclear expression factor,expressed in tobacco; human erythropoietin, expressed in tobacco; humanenkephalins, expressed in Arabidopsis; human epidermal growth factor,expressed in tobacco; human interferon-alpha, expressed in rice, turnip(Brassica rapa); human β-interferon, expressed in tobacco; human serumalbumin, expressed in tobacco; human α, β hemoglobin, expressed intobacco; human homotrimeric collagen, expressed in tobacco; humanα-1-antitrypsin, expressed in rice; human aprotinin, expressed in maize;human lactoferrin, expressed in potato; angiotensin-converting enzyme,expressed in tobacco, tomato; α-tricosanthin from TMV-U1, expressed inNicotiana bethamiana; and glucocerebrosidase, expressed in tobacco(Daniell et al., Trends Plant Sci. 6:219-226, 2001).

Enzymes with industrial applications which can be expressed bytransgenic plants currently include α-amylase, expressed in tobacco;phytase, expressed in alfalfa, tobacco; cellulose, expressed in alfalfa,potato, tobacco; manganese peroxidase, expressed in alfalfa, tobacco;β-(1,4) xylanase, expressed by tobacco, canola; β-(1,3) glugcanase,expressed by tobacco, barley; and glucuronidase, expressed in maize(Rishi et al., J Plant Biochemistry & Biotechnology Vol. 10:1-12, 2001).

Biopolymers with industrial applications that can be generated bytransgenic plants currently include poly-3-hydroxyalkanoates (PHAs).PHAs show material properties that are similar to some common plasticssuch as polypropylene. Besides being biodegradable, PHAs are recyclablelike petrochemical thermoplastics. Production of PHAs has been reportedin transgenic Arabidopsis thaliana plants (Poirier et al, Science 256:520-523, 1992), cotton (U.S. Pat. No. 5,602,321) and radish (Slater etal., Nature Biotechnology 17: 1011-1016, 1999).

A suitable exogenous polynucleotide sequence, according to the teachingof the present invention, may also encode a polypeptide capable ofconferring plant resistance or tolerance to a biotic stress such as, butnot limited to, pest or disease resistance. Presently known polypeptidescapable of conferring pest or disease resistance in transgenic plantsinclude, for example, insecticidal Bacillus thuringiensis (Bt)crystalline proteins, fungal cell-wall degrading enzymes such aschitinases and glugcanases (such as described in U.S. Pat. No.6,521,4350), pathogenesis-related proteins (such as described byMcDowell and Woffenden, Trends Biotechnol. 21:178-83, 2003),antimicrobial peptides (such as described in U.S. Pat. No. 6,600,090)and proteins involved in the signal transduction cascade leading tosystemic acquired resistance in plants (such as described in U.S. Pat.No. 6,091,004).

A suitable exogenous polynucleotide sequence may also encode apolypeptide capable of conferring plant resistance or tolerance to anabiotic stress such as, but not limited to, drought, salinity, extremetemperature, flood, frost, malnutrition, toxic pollution, UV irradiationand a mechanical injury. Polypeptides capable of conferring planttolerance to an abiotic stress in transgenic plants are described in,for example, U.S. Pat. Nos. 5,965,705, 6,613,919, 6,563,019 and Kasugaet al. (Nat Biotechnol. 17: 287-91, 1999).

A suitable exogenous polynucleotide sequence may also encodenutritionally valuable polypeptides such as described, for example, bySevenier et al. (J Am Coll Nutr. 21:199-204, 2002).

As is mentioned hereinabove, infecting the plant harboring the exogenouspolynucleotide sequence with a virus capable of suppressing genesilencing enhances the expression of exogenous polynucleotide sequencein the plant. There are currently over 500 known viruses capable ofinfecting almost all plants; examples of which are described in the website http://image.fs.uidaho.edu/vide/. A suitable virus, according tothe teaching of the present invention, is capable of suppressing genesilencing and capable of systemically infecting the plant, i.e.,establishing and propagating throughout the plant body. Examples ofviruses capable of systemically infecting crop plants are provided inTable 1 which follows. TABLE 1 Plant family Poty- Tobamo- Cucumo Nepo-Gemini ▾ virus virus virus virus virus Solanacea (e.g., PVY RMV CMVCGMV, TYLCV tomato, tobacco) ArMV Cereals e.g., corn, MDMV, RMV MSVwheat, rice) SCMV Soybean SMV, CMV ArMV CMV Cucurbits (e.g., ZYMV RMVCMV ArMV SLCV cucumber, melon, squash, pumkin, watermelon)Abbreviations:CMV, cucumber mosaic virus;CGMV, cassava green mottle nepovirus;ArMV, Arabis mosaic nepovirus;PVY, potato virus Y;ZYMV, zuccini yellow mosaic virus;SMV, soybean mosaic potyvirus;BCMV, bean common mosaic potyvirus;MDMV, Maize dwarf mosaic potyvirus;MSV, maize streak monogeminivirus;SCMV, sugarcane mosaic potyvirus;RMV, ribgrass mosaic tobamovirus;TYLCV, tomato yellow leaf curl bigeminivirus;SLCV, squash leaf curl virus.

Preferably, the virus is avirulent and thus is incapable of causingsevere symptoms such as reduced growth rate, mosaic, ring spots, leafroll, yellowing, streaking, pox formation, tumor formation and pitting.A suitable avirulent virus may be a naturally occurring avirulent virusor an artificially attenuated virus. Virus attenuation may be effectedby using methods well known in the art including, but not limited to,sub-lethal heating, chemical treatment or by directed mutagenesistechniques such as described, for example, by Kurihara and Watanabe(Molecular Plant Pathology 4:259-269, 2003), Gal-on et al. (1992),Atreya et al. (1992) and Huet et al. (1994).

Suitable virus strains can be obtained from available sources such as,for example, the American Type culture Collection (ATCC) or by isolationfrom infected plants. Isolation of viruses from infected plant tissuescan be effected by techniques well known in the art such as described,for example by Foster and Tatlor, Eds. “Plant Virology Protocols: FromVirus Isolation to Transgenic Resistance (Methods in Molecular Biology(Humana Pr), Vol 81)”, Humana Press, 1998. Briefly, tissues of aninfected plant believed to contain a high concentration of a suitablevirus, preferably young leaves and flower petals, are ground in a buffersolution (e.g., phosphate buffer solution) to produce a virus infectedsap which can be used in subsequent inoculations.

Isolated viruses or virus-infected sap can be freeze-dried (lyophilized)in order to improve viral preservation in storage, using methods wellknown in the art such as described, for example, by Rowe et al.(Cyrobiology 8: 153-72, 1971) and by Rightsel et al. (Cryobiology3:423-31, 1967).

The virus of the present invention can be, for example, a strain or anisolate of a potyvirus (e.g., potato virus Y, PVY; tobacco etch virus,TEV), a cucumovirus (e.g., cucumber mosaic virus, CMV), a comovirus(e.g., cowpea mosaic virus, CpMV), a geminivirus (e.g., cassaya mosaicvirus, ACMV), a nepovirus (e.g., nandina virus X, NMV; viola mosaicvirus, VMV), a tomavirus (e.g., tobacco mosaic virus, TMV) and atobavirus (e.g., tobacco black ring virus, TBSV).

The virus can be administered to a plant per se or as part (activeingredient) of an inoculant formulation. A suitable inoculantformulation may include the virus and an acceptable carrier such as astabilizer.

The term “stabilizer” used herein refers to any inert substance which iscapable of increasing virus stability in storage. Suitable stabilizersmay include sugars such as sucrose, raffinose, glucose and trehalose, ora composition such as described, for example, in U.S. Pat. Nos.6,290,967, 6,544,769, 4,186,195, 4,147,772, 4,000,256, 3,783,098.

Techniques for formulation and inoculation of viruses to plants may befound in Foster and Taylor, eds. “Plant Virology Protocols: From VirusIsolation to Transgenic Resistance (Methods in Molecular Biology (HumanaPr), Vol 81)”, Humana Press, 1998; Maramorosh and Koprowski, eds.“Methods in Virology” 7 vols, Academic Press, New York 1967-1984; Hill,S. A. “Methods in Plant Virology”, Blackwell, Oxford, 1984; Walkey, D.G. A. “Applied Plant Virology”, Wiley, New York, 1985; and Kado andAgrawa, eds. “Principles and Techniques in Plant Virology”, VanNostrand-Reinhold, New York.

To facilitate use, the inoculant formulation is preferably packaged in asealed container labeled for use as an inoculant along with informationindicating suitable target plants, plant stage most suitable forinoculation and recommended application rates.

Inoculant formulations suitable for use in context of the presentinvention include formulations wherein the viruses are contained in anamount effective to achieve the intended purpose. For any preparationused in the methods of the invention, the effective amount or dose canbe estimated initially from small-scale growth-chamber or greenhousetrials. Such information can be used to more accurately determine usefuldoses in commercial large-scale applications. Dosages necessary toachieve the desired effect will depend on the virus specificcharacteristics and the specific plant growth stage and environmentalcharacteristic. The method of the present invention is effected asfollows: A plant genetically modified to express a molecule of interest(e.g., an antibody, a vaccine, a therapeutic polypeptide an industrialenzyme, a polypeptide conferring stress resistance or tolerance, or anutritionally valuable polypeptide) is inoculated with a suitable virusstrain by applying viral containing sap or any other viral preparationto the surface of plant tissue, preferably leaves, which was previouslydusted with an abrasive such as carborundum. Application of the sap ispreferably made by gently rubbing the leaves with a pad dipped in thesap, with a finger, a glass spatula, a painter's brush, or with a smallsprayer. In successful inoculation, the virus enters the plant cellsthrough the wounds made by the abrasive or through other opening andinitiates an infection.

For high volume inoculations, the virus is preferably administered tothe plant by using a mechanized plant inoculation instrument such asdescribed, for example, by Gal-on et al. (J. Gen. Virology 3223-3227,1995) and in U.S. Pat. No. 6,644,341.

Once expression of the polypeptide reaches suitable levels thepolypeptide may be extracted from plant tissues. General methods ofextracting polypeptides from plant tissues are well known in the artsuch as described, for example, by Roe, S., Ed. (“Protein Purification:a Practical Approach”, Oxford University Press, 2001). Preferably,target molecules such as antibodies, vaccines and therapeuticpolypeptides are extracted and purified from plant tissues usingprocedures such as described by Cunningham and Porter, Eds.,“Recombinant Protein Production in Plants: Production and Isolation ofClinically Useful Compounds”, Humana Press, 1998; Fischer et al.,Biotechnol. Appl. Biochem. 30:101-108, 1999; and Seon et al., J. PlantBiotechnology 4: 95-101, 2002. Procedures of extracting biopolymers fromplant tissues are described in Doi Y. and Steinbuchel, Eds. “BiopolimersVolume 4”, Willey-VCH, 2002.

Since plants expressing a detectable transgene can be used to qualifyand quantify gene silencing suppression of a specific viral isolate, thepresent invention also envisages a method of identifying viral isolateswhich are capable of effectively suppressing gene silencing in plants.

Such a method is effected by inoculating a plurality of transgenicplants expressing a detectable polypeptide (e.g., reporter polypeptide)with a plurality of virus isolates or strains, using inoculation methodssuch as described hereinabove. Following inoculation, an infected plantthat exhibits a substantially higher level of reporter polypeptideexpression than a non-infected similar transgenic plant is selected.Preferably, the selected plant does not exhibit severe symptoms such asmosaic, ring spots, leaf roll, yellowing, streaking, pox formation,tumor formation, pitting and stunting. The virus isolate or straininfecting the selected plant is thereby isolated and identified as agene silencing suppressing agent.

Infected plants may be analyzed to determine the effect of virusinoculation on the expression of exogenous polynucleotide sequences bymeasuring the levels of the reporter polypeptide or its transcribedmRNAs.

Reporter polypeptide expression in plant tissues can be quantitativelyanalyzed using standard protein detection assays which are well known inthe art such as, for example, enzyme-linked immuno-sorbent assay (ELISA)and Western blot analysis; or by using fluorescent based assays in thecase of a fluorescent reporter (e.g., GFP), or a substrate based assayin the case of an enzyme reporter (e.g., luciferase). The level oftranscribed mRNAs can be analyzed by hybridization-based assays such as,for example, reverse-transcription polymerase chain-reaction (RT-PCR)and Northern blot analysis (see, for example, Clark, Ed., PlantMolecular Biology: A Laboratory Manual, Springer-Verlag, Berlin, 1997;Glick and Thompson, Eds., “Methods in Plant Molecular Biology andBiochemistry, CRC Press, 1993; Dashek, W. V., Eds., “Methods in PlantPathology and Molecular Biology, CRC Press, 1997; Weissbach andWeissbach, Methods for Plant Molecular Biology, Academic Press, 1988;and in the web site http://www.protocol-online.org/prot/Plant Biology/).

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions illustrate the invention in a non-limiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., Ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., Ed. (1994); “Current Protocols in Immunology” Volumes I-IIIColigan J. E., Ed. (1994); Stites et al. (eds), “Basic and ClinicalImmunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994);Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W.H. Freeman and Co., New York (1980); available immunoassays areextensively described in the patent and scientific literature, see, forexample, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578;3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521;“Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic AcidHybridization” Hames, B. D., and Higgins S. J., eds. (1985);“Transcription and Translation” Hames, B. D., and Higgins S. J., eds.(1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “ImmobilizedCells and Enzymes” IRL Press, (1986); “A Practical Guide to MolecularCloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317,Academic Press; “PCR Protocols: A Guide To Methods And Applications”,Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategiesfor Protein Purification and Characterization—A Laboratory CourseManual” CSHL Press (1996); all of which are incorporated by reference asif fully set forth herein. Other general references are providedthroughout this document. The procedures therein are believed to be wellknown in the art and are provided for the convenience of the reader.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below.

Example 1 Increasing Expression of Transgenic GFP in Tobacco by anAvirulent PVY Strain

Materials and Methods:

Plants and growth conditions: Transgenic Nicotiana tabacum R3 and R6carrying the Green Fluorescence Protein (GFP) gene and wild type N.tabacum SR were planted in peat-based growth mix and grown in a growthchamber under day/night conditions of 14 hr at 24° C./10 hr at 20° C.

Virus: Potato Virus Y (PVY) strain N605 described by Jakab et al.(Journal of General Virology 78:3141-3145, 1997) was used.

Virus inoculation: Tobacco leaf tissue infected with PVY N₆O₅ was groundby pestle and mortar in a 1:4 dilution of 0.05 M ice-cold phosphatebuffer, pH 7.0, to produce a virus infected sap. The sap was rubbed witha cotton swab onto carborundum-dusted leaves of virus free tobaccoplants.

RT-PCR analysis: Reverse Transcription Polymerase Chain Reaction(RT-PCR) was performed using SuperScript II according to themanufacturer instructions (Invitrogen Life Technologies; Huang, L. etal., Focus 22:3, 2000) using the following primers: 5′-GAT CCA GCA AAGGGG TAT TCA GCA TA (SEQ ID NO: 1) and 5′-TCT GCA TCA TGN ACR TCA GG (SEQID NO: 2).

Fluorometer analysis: Fluorescence specific activity was measured inplant tissue using the procedure described by Remens et al. (1999).

Results:

No visual symptoms were observed in any of the PVY infected plants,while RT-PCR analysis on the leaves 2 to 4 weeks after inoculationindicated systemic transmission of PVY within infected plants.

As illustrated in FIG. 1, the fluorescence specific activity measured innon-inoculated GFP-transgenic plants was 100% higher than innon-inoculated wild-type plants (0.6 and 0.3 R.U/mg protein inGFP-transgenic and wild-type plants, respectively), indicating asubstantially enhanced GFP expression in the transgenic plants.

PVY infection did not alter the level of fluorescence measured inwild-type plants. However, PVY-infected GFP transgenic plants exhibitedsignificantly higher level of fluorescence, as compared withnon-infected GFP transgenic plants (1.05 and 0.6 R.U/mg protein, inPVY-infected and noninfected transgenic plants, respectively; p<0.001).

These results indicate that inoculation of transgenic tobacco plantswith an avirulent PVY strain can substantially enhance the expression ofexogenous polynucleotide sequences.

Example 2 Increasing Expression of Transgenic mRNA in Arabidopsis by anAvirulent CMV Strain

Materials and Methods:

Plants and growth conditions: Rab7 transgenic Arabidopsis thaliana andwild type A. thaliana Columbia were planted in peat-based growth mix andmaintained under 16 h light at 24° C. and 8 h dark at 22° C.

Virus: Cucumber Mosaic Virus (CMV) Banana strain as described by Gafnyet al. (Phytoparasitica 24:49-56, 1996) was used.

Virus inoculation: Leaf tissue of banana plants infected with CMV Bananastrain was grounded by pestle and mortar in a 1:4 dilution of 0.05 M icecold phosphate buffer, pH 7.0, to produce a virus infected sap. The sapwas rubbed with a cotton swab onto carborundum-dusted leaves of virusfree A. thaliana plants.

RT-PCR analysis: The PCR procedure was performed as described in example1 above using the following primers: 5′-GAG, CGG, TCA, CAA, GAG, AGT, AG(SEQ ID NO: 3) and 5′-GGA, AAT, CAC, ACC, ACC, ACT, TA (SEQ ID NO: 4).

Northern blot analysis: RNA was blotted into Hybond N+ membranefollowing the protocol recommended by the manufacturer (AmershamPharmacia Biotec).

Results:

As illustrated in FIG. 2, the inoculation of transgenic Arabidopsisthaliana with an infectious but avirulent CMV (Banana strain) resultedin 123-139% increase in the level a transgene transcribed mRNA, ascompared with non-inoculated control.

These results indicate that inoculation of transgenic Arabidopsis plantswith an avirulent CMV strain can substantially enhance the expression ofexogenous polynucleotide sequence mRNA.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications, patents, patent applicationsand sequences identified by their accession numbers mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent, patent application or sequence identified by theiraccession number was specifically and individually indicated to beincorporated herein by reference. In addition, citation oridentification of any reference in this application shall not beconstrued as an admission that such reference is available as prior artto the present invention.

REFERENCES Additional References are Cited Hereinabove

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1. A method of enhancing an expression of an exogenous polynucleotidesequence in a plant, comprising administering to the plant a virusselected capable of suppressing gene silencing in said plant, therebyenhancing the expression of the exogenous polynucleotide sequence insaid plant.
 2. The method of claim 1, wherein said virus is asystemically infectious virus.
 3. The method of claim 1, wherein saidvirus is an avirulent virus.
 4. The method of claim 1, wherein saidvirus is a mechanically transmitted virus.
 5. The method of claim 1,wherein said administering is effected by using an inoculation gun.
 6. Amethod of identifying a gene silencing agent, comprising: (a)inoculating a plurality of transgenic plants with a plurality of virusisolates or strains thereby generating a plurality of infected plants;and (b) selecting a plant from said infected plants which exhibits asubstantially higher level of exogenous polynucleotide sequenceexpression than a non-infected similar transgenic plant, therebyidentifying the virus isolate or strain infecting said plant as the genesilencing agent.
 7. The method of claim 6, wherein step (a) furtherincludes selecting plants which do not exhibit severe symptoms.
 8. Themethod of claim 6, wherein step (a) is effected by administering saidvirus isolates or strains suspended in a buffer solution supplementedwith an abrasive material onto foliage of said transgenic plants.
 9. Themethod of claim 6, wherein said symptoms are selected from the groupconsisting of mosaic, ring spots, leaf roll, yellowing, streaking, poxformation, tumor formation, pitting and stunting.
 10. The method ofclaim 6, wherein said exogenous polynucleotide sequence expression isquantified by an exogenous polynucleotide sequence transcribed mRNAlevel.
 11. The method of claim 6, wherein said exogenous polynucleotidesequence expression is quantified by said exogenous polynucleotidesequence encoded polypeptide level.
 12. A method of producing a moleculeof interest, comprising: (a) administering to a plant a virus selectedcapable of suppressing gene silencing in said plant; and (b) extractingthe molecule of interest being expressed in said plant, therebyproducing said molecule of interest.
 13. The method of claim 12, whereinsaid molecule of interest is selected from the group consisting of anantibody, a vaccine, a therapeutic polypeptide, an industrial enzyme anda biopolymer.
 14. The method of claim 12, wherein said molecule ofinterest is a polypeptide capable of conferring resistance or toleranceto biotic stress.
 15. The method of claim 12, wherein said molecule ofinterest is a polypeptide capable of conferring resistance or toleranceto abiotic stress.
 16. The method of claim 12, wherein said molecule ofinterest is a nutritionally valuable polypeptide.
 17. The method ofclaim 12, wherein said virus is a systemically infectious virus.
 18. Themethod of claim 1, wherein said virus is an avirulent virus.
 19. Themethod of claim 12, wherein said virus is a mechanically transmittedvirus.
 20. The method of claim 12, wherein said administering iseffected by using an inoculation gun.
 21. An article-of-manufacturing,comprising a container including a virus selected capable of suppressinggene silencing in a plant, and a packaging material identifying saidvirus for use in innoculating said plant.
 22. The method of claim 21,wherein said virus is a systemically infectious virus.
 23. The method ofclaim 21, wherein said virus is an avirulent virus.
 24. The method ofclaim 21, wherein said virus is a mechanically transmitted virus. 25.The method of claim 21, wherein said virus is lyophilized.